1. Field of Invention
The present invention relates to polymerase chain reaction (PCR) assays for a quantitation of human DNA, and more particularly multiplex PCR for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA.
2. Description Of the Related Art
Forensic DNA specimens are commonly matched to alleged criminal suspects in modern law enforcement using human identification systems validated according to the DNA Advisory Board's (DAB) Quality Assurance Standards. These DNA testing systems typically involve the amplification of highly polymorphic short tandem repeats (STR's) by polymerase chain reaction (PCR). AmpF/STR® Profiler Plus and COfiler®, Powerplex®, Powerplex-Y, and Y-PLEX™ 12 are examples of commonly used multiplex systems for genotyping polymorphic STRs residing in the human nuclear (nDNA) and male Y-chromosome DNA. In general, these systems require about 0.1 to 2.0 ng of DNA template for analysis (W. E. Frank et al., J Forensic Sci 46 (2001) 642-646). When DNA evidence is limited, analysis of human mitochondrial DNA (mtDNA) targets is often employed because of the high copy number of mitochondria and mtDNA molecules in each cell (D. Bogenhagen et al., J Biol Chem 249 (1974) 7991-7995). In some cases, DNA evidence obtained from criminal investigations or forensic anthropology studies may be degraded and/or contain non-human contaminants. Consequently, an essential precursor to standard forensic analyses is the sensitive quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA from complex biomaterials.
Commercially available products for human DNA quantitation include AluQuant (Promega Corp.), Quantifiler™ human and Quantifiler™ Y (Applied Biosystems, Inc.). In addition, several real-time PCR assays have been reported for nDNA quantitation (J. C. Fox et al., Biotechniques 34 (2003) 314-322, J. A. Nicklas et al., J Forensic Sci 48 (2003) 936-944, E. Sifis et al., J Forensic Sci 47 (2002) 589-592, J. A. Walker et al., Anal Biochem 315 (2003) 122-128, J. A. Nicklas et al., J. Forensic Sci. 48 (2003) 282-291,) and for the simultaneous quantitation of nDNA and mtDNA (A. Alonso et al., Forensic Science International 139 (2004) 141-149, and H. Andreasson et al., Biotechniques 33 (2002) 402-411).
There are limitations to these previously reported methods. Many of these systems are not human specific and amplify both human and non-human primate DNA (J. C. Fox et al., Biotechniques 34 (2003) 314-322, J. A. Nicklas et al., J Forensic Sci 48 (2003) 936-944, M. E. Sifis et al., J Forensic Sci 47 (2002) 589-592, J. A. Nicklas et al., J. Forensic Sci. 48 (2003) 282-291, and H. Andreasson et al., Biotechniques 33 (2002) 402-411). Some methods are not target specific in that the mtDNA assay also amplifies a product from the nuclear genome (H. Andreasson et al., Biotechniques 33 (2002) 402-411), or is designed in the hyper-variable region (HV) of the mitochondrial genome rather than in the conserved region, thereby increasing the probability of target mismatches and inaccurate quantitation (A. Alonso et al., Forensic Science International 139 (2004) 141-149). Most of these systems are not multiplex compatible (Quantifiler™, Quantifier Y, AluQuant) (J. C. Fox et al., Biotechniques 34 (2003) 314-322, J. A. Nicklas et al., J Forensic Sci 48 (2003) 936-944, M. E. Sifis et al., J Forensic Sci 47 (2002) 589-592 and J. A. Walker et al., Anal Biochem 315 (2003) 122-128), resulting in the possible depletion of valuable DNA specimen prior to subsequent analyses. Most importantly, no system is currently available that meets all of these criteria, human specificity, target specificity, high sensitivity and multiplex compatible.